Parathyroid Hormone-Related Peptide
Overview: The physiological role of PTHrP isn’t completely understood, but its functions can be divided into 5 categories: * Transepithelial calcium transport, particularly in the kidney and mammary gland * Smooth muscle relaxation in the uterus, bladder, gastrointestinal tract, and arterial wall * Regulation of cellular proliferation * Cellular differentiation and apoptosis of multiple tissues * As an indispensable component of successful pregnancy and fetal development (embryonic gene deletion is lethal in mammals)3 The PTHrP can be very useful for diagnostic work-up of patients with suspected hypercalcemia of malignancy and/or of unknown origin. Approaches to inhibit its expression or its effects by malignant cells hold promise for treating the hypercalcemia and osteolysis associated with some cancers4. Construct Used For Expression: Recombinant human parathyroid hormone-related protein can be synthetically produced by bacterial cells, mainly E.coli. In order for the human parathyroid hormone to be expressed within the E.coli cells, the recombinant vector pTYB1-hPTHrP (1–139) is introduced into the protease-deficient E. coli strain ER2566. The culture is then grown in a broth with ampicillin, and the expression of recombinant hPTHrP is induced by adding isopropyl-d-thiogalactopyranoside (IPTG)2. Looking at this in more detail, the cDNA encoding PTHrP(1-139) is ligated into the Multiple Cloning Site (MCS) of the pTYB1 plasmid. It’s inserted into the Ndel and SapI sites of the MCS because the Ndel site allows the recombinant PTHrP to have its natural N-terminus and the SapI site allows the PTHrP to fuse its C-terminus to the N-terminal cysteine of intein so that when it is cleaved it won’t have any vector-derived amino acid residues. The role of the intein is to act as a protein catalyze for the protein slicing that occurs. Once the cDNA is inserted into the plasmid DNA of the E.coli cells, they are grown within an antibiotic and IPTG which forces the bacterial cell to transcribe the PTHrP along with the antibiotic resistant gene associated with the inserted cDNA2. Purification: Once the bacterial cells are able to produce a sufficient amount of the PTHrP is needs to be extracted and purified. The bacterial cells are first put through a centrifuge, then re-suspended in ice-cold column buffer (20 mM Tris–HCl, pH 8.0; 500 mM NaCl; 0.1 mM EDTA; 0.1% Triton X-100), and undergo sonication in order for the cells to be lysed. Once the cells are lysed, they are put back into the centrifuge in order to remove the cellular debris created by lysis of the cells, and then loaded onto a chitin column to be washed with column buffer at a slow rate. After this step, the column is then flushed with 3 volumes of column cleavage buffer (20 mM Tris–HCl, pH 8.0; 1 M NaCl; 0.1 mM EDTA) which contains DTT. After being allowed to sit over night, the PTHrP is eluted by adding a cleavage buffer without DTT and concentrated using a Centricon YM-3 filtration unit2. Resources: 1. www.Wikipedia.com 2. Single-Column Purification and Bio-Characterization of Recombinant Human Parathyroid Hormone-Related Protein by Wu C, Seitz PK, and Falzon M. PMID:11162900 3. Parathyroid Hormone-Related Peptide by www.mayomedicallaboratories.com 4. PTH-Related Peptide (PTHrP) in Hypercalcemia by Gregory Mundy and James Edwards 5. Ideal Protein Purification by igem.org